Phorbol dibutyrate-induced megakaryocytic differentiation increases susceptibility of K562 cells to SA11 rotavirus infection

2001 ◽  
Vol 146 (9) ◽  
pp. 1831-1840 ◽  
Author(s):  
G. M. Sanders ◽  
M. J. Hewish ◽  
B. S. Coulson
Keyword(s):  
K562 Cells ◽  
Megakaryocytic Differentiation ◽  
Rotavirus Infection ◽  
Phorbol Dibutyrate

Membrane type sialidase inhibits the megakaryocytic differentiation of human leukemia K562 cells

2008 ◽  
Vol 1780 (5) ◽  
pp. 757-763 ◽  
Author(s):  
Un-Ho Jin ◽  
Ki-Tae Ha ◽  
Kyung-Woon Kim ◽  
Young-Chae Chang ◽  
Young-Coon Lee ◽  
...  
Keyword(s):  
K562 Cells ◽  
Human Leukemia ◽  
Megakaryocytic Differentiation ◽  
Membrane Type ◽  
Human Leukemia K562 Cells

scholarly journals Effects of THAP11 on Erythroid Differentiation and Megakaryocytic Differentiation of K562 Cells

PLoS ONE ◽  
2014 ◽  
Vol 9 (3) ◽  
pp. e91557 ◽  
Author(s):  
Xiang-Zhen Kong ◽  
Rong-Hua Yin ◽  
Hong-Mei Ning ◽  
Wei-Wei Zheng ◽  
Xiao-Ming Dong ◽  
...  
Keyword(s):  
K562 Cells ◽  
Erythroid Differentiation ◽  
Megakaryocytic Differentiation

PHD Domain Deletion Mutations of ASXL1 Promote Myeloid Leukemia Transformation Through Epigenetic Dysregulation and Inhibit Megakaryocytic Differentiation Through the Inactivation of FOSB in K562 Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2393-2393 ◽  
Author(s):  
Rabindranath Bera ◽  
Der-Cherng Liang ◽  
Ming-Chun Chiu ◽  
Ying-Jung Huang ◽  
Sung-Tzu Liang ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Blast Crisis ◽  
K562 Cells ◽  
Specific Cell ◽  
Wild Type ◽  
Megakaryocytic Differentiation ◽  
Deletion Mutations ◽  
Phd Domain

Abstract Abstract 2393 Somatic mutations of ASXL1 gene have been described in patients with myeloid malignancies and were associated with inferior outcomes. ASXL1 mutations have also been detected in myeloid blast crisis of chronic myeloid leukemia (CML) patients. The mechanisms of acute myeloid leukemia (AML) transformation and functional role of ASXL1 mutations in the leukemogenesis remain to be determined. Recently, we identified PHD domain deletion mutations (R693X and L885X) in patients with CML in myeloid blast crisis and/or AML with minimal differentiation (M0). In the present study, we aimed to investigate the role of PHD domain deletion mutations in the pathogenesis of AML transformation. The K562 cells carrying Philadelphia chromosome, serves as a model to study the molecular mechanisms associated with leukemogenesis. Our result showed that R693X/L885X mutations inhibited PMA-treated megakaryocytic differentiation with the change of physiological characteristic features and suppressed the induction of CD61, a specific cell surface marker of megakaryocytes. We also found that FOSB, a member of Fos family of AP-1 transcription factors was down-regulated in K562 cells expressing R693X and L885X compared to wild-type ASXL1 during PMA-mediated megakaryocytic differentiation. Examination of intracellular signaling pathways showed that the mutant ASXL1 protein prevented PMA-induced megakaryocytic differentiation through the inactivation of ERK, AKT and STAT5 which are required for differentiation. Further, ASXL1 depletion by shRNA in K562 cells led to enhanced cell proliferation, increased colony formation and impaired PMA-mediated differentiation. Previous studies in Drosophila had revealed that Asxl forms the protein complexes of both Trithorax and Polycomb groups that are required for maintaining chromatin in both activated and repressed transcriptional states. By using Western blot analysis, we demonstrated that PHD domain deletion mutations of ASXL1 significantly suppressed the transcriptionally repressive mark H3K27 trimethylation, however no effect on methylated H3K4 (H3K4me2 and H3K4me3), an active histone mark in K562 cells. Co-immunoprecipitation analysis revealed that wild-type, but not PHD domain deletion mutations of ASXL1 interact with EZH2, a member of the polycomb repressive complex 2 (PRC2). Importantly, PHD deletion mutations or downregulation of ASXL1 resulted in the suppression of EZH2 in K562 cells. Our study demonstrated that PHD deletion mutations of ASXL1 resulted in a loss-of-function which exhibited direct effects on the proliferation and differentiation and also proposed a specific role for ASXL1 in epigenetic regulation of gene expression in K562 cells. Disclosures: No relevant conflicts of interest to declare.

VASP Protein, a Binding Partner Of BCR-ABL and FAK, May Be Implicated In The Chronic Myeloid Leukemia Phenotype

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5162-5162
Author(s):  
Vanessa Aline Bernusso ◽  
João Agostinho Machado-Neto ◽  
Fernando V Pericole ◽  
Karla Priscila Vieira ◽  
Adriana Silva Santos Duarte ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
Fold Increase ◽  
Imatinib Treatment ◽  
Gene Expressions ◽  
Megakaryocytic Differentiation ◽  
Healthy Donors

Abstract Background VASP (vasodilator-stimulated phosphoprotein) and Zyxin are actin regulatory proteins that control cell-cell adhesion. Zyxin directs actin assembly by interacting and recruiting VASP to specific sites of adhesion. The phosphorylation of VASP modifies their activity in cell-cell junctions. PKA phosphorylates VASP at serine 157 regulating VASP cellular functions. VASP is a substrate of BCR-ABL oncoprotein and is tyrosine-phosphorylated in leukemic cells. However, the function of VASP and Zyxin in hematopoietic cells, in the BCR-ABL pathway and its participation in chronic myeloid leukemia (CML) remains unknown. Aims To analyze VASP and Zyxin expression in bone marrow cells from CML patients and healthy donors, as well the involvement of these proteins in hematopoietic cell differentiation and in the BCR-ABL signaling pathway. Materials and Methods VASP and Zyxin expression and phosphorylation were studied in bone marrow samples from 29 individuals (5 healthy donors, 5 CML patients at diagnosis, 16 CML patients responsive to treatment with tyrosine kinase inhibitors (ITK) and 3 CML patients resistant to ITK). One patient was analyzed at diagnosis and after ITK response. VASP or Zyxin silencing was performed by shRNA-lentiviral delivery in K562 cell line, an appropriated shControl was used. ShControl, shVASP and shZyxin K562 cells were induced to megakaryocytic differentiation with 20nM of PMA (phorbol myristate -13 -12 acetate) during 4 days and CD61 expression, a marker for maturing megakaryocytes, was verified by flow cytometry. During megakaryocytic differentiation, VASP and Zyxin gene expressions were evaluated by quantitative PCR; protein expression and activation were determined by Western blotting. Effector proteins of proliferation, apoptosis and adhesion in the BCR-ABL signaling pathway were analyzed in cells silenced for VASP or Zyxin. The interaction of VASP and BCR-ABL or FAK was evaluated by co-immunoprecipiation. Results Healthy donors showed p-VASP ser157 expression, in contrast to CML patients at diagnosis who did not present phospho-VASP ser157. After Imatinib treatment CML patients restored VASP phosphorylation however resistant patients maintained this absence. Zyxin showed the same expression in patients and healthy donors. During Imatinib treatment of K562 cells, phospho-VASP ser157 expression was increased and its interaction with BCR-ABL protein was reduced. VASP and Zyxin gene expressions were upregulated during megakaryocyte differentiation of K562 cells (8.7-fold increase, P=0.0115, and 3.6-fold increase, P=0.015, respectively). VASP and Zyxin protein expressions were increased during megakaryocytic differentiation, including the active form of these proteins (p-VASP ser157 and p-Zyxin ser142). VASP silencing in K562 cells resulted in a 40% decrease of CD61 expression at the end of the megakaryocytic differentiation (P<0.05). In addition, VASP and Zyxin silencing resulted in a decrease of BCL-2 and BCL-XL proteins. VASP binds to FAK, an adhesion effector protein of the BCR-ABL pathway, and it´s silencing resulted in a decreased phosphorylation of FAK y925. Conclusions In BCR-ABL cells, VASP and Zyxin modulated anti-apoptotic proteins and megakaryocytic differentiation. Hence, the altered expression of VASP activity in CML patients may contribute to the pathogenesis of the disease, affecting cellular differentiation or leukemic cell adhesion. Disclosures: No relevant conflicts of interest to declare.

Negative regulation of globin gene expression during megakaryocytic differentiation of a human erythroleukemic cell line

1991 ◽  
Vol 11 (7) ◽  
pp. 3528-3536
Author(s):  
N L Lumelsky ◽  
B G Forget
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Negative Regulation ◽  
Myristate Acetate ◽  
Globin Mrna ◽  
Megakaryocytic Differentiation ◽  
Gamma Globin ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

The human erythroleukemic cell line K562 was used as a model for analysis of the mechanisms responsible for alterations in gene expression during differentiation. K562 cells normally synthesize fetal hemoglobin (gamma-globin), but treatment with tumor-promoting phorbol esters (phorbol myristate acetate and tetradecanoyl phorbol acetate) results in the loss of the erythroid phenotype of the cells and causes a shift toward a megakaryocytic phenotype. This shift involves markedly decreased production of fetal hemoglobin and de novo synthesis of a number of proteins specific for megakaryocytes. The results of this work indicate that negative regulation of fetal hemoglobin during megakaryocytic differentiation of K562 cells occurs at the level of down regulation of gamma-globin mRNA accumulation. This effect consists of at least two components: reduction in the rate of transcription of the gamma-globin gene and decrease in stability of the normally very stable gamma-globin mRNA. We have developed two assay systems that permit investigation of the transcriptional and posttranscriptional effects of phorbol myristate acetate independently from each other. These assay systems make use of a heterologous reporter gene for the transcriptional analysis and a marked gamma-globin gene for the analysis of mRNA stability. The DNA sequences located in the 3' flanking region of the A gamma-globin gene were found to be responsible for the decrease in transcription rate.

Crispr/Cas9-Induced DNMT3A Mutations in the K562 Human Leukemic Cell Line As a Model of DNMT3A-Mutated Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2704-2704 ◽  
Author(s):  
Lauren G. Banaszak ◽  
Valentina Giudice ◽  
Xin Zhao ◽  
Zhijie Wu ◽  
Kohei Hosokawa ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genomic Instability ◽  
Malignant Transformation ◽  
Cancer Cell ◽  
Research Funding ◽  
K562 Cells ◽  
Human Cells ◽  
Genomic Integrity ◽  
Megakaryocytic Differentiation ◽  
Apoptotic Activity

Abstract Background:DNA methyltransferase 3A(DNMT3A) is a member of the DNA methyltransferase family primarily involved in de novo gene methylation. Mutations in DNMT3A are associated with a wide range of hematological malignancies, most frequently acute myeloid leukemia (AML). DNMT3A mutations are thought to produce a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. Mutations in DNMT3A often coexist with secondary lesions in leukemia-related genes such as NPM1 and FLT3 (Ley T et al., N Engl J Med, 2010). Furthermore, healthy individuals harboring DNMT3A-driven clonal hematopoiesis are at increased risk of future hematologic malignancies and all-cause mortality (Jaiswal S et al., N Engl J Med, 2014). Despite these important clinical associations, the mechanisms by which DNMT3A mutations contribute to malignant transformation have not been well-defined. Dnmt3a-knockout (KO)mouse hematopoietic stem cells (HSCs) preferentially self-renew rather than undergo differentiation, leading to their accumulation in the bone marrow (Challen GA et al., Nat Genet, 2011). DNMT3A loss has also been shown to drive hypomethylation and subsequent activation of leukemia-related genes (Lu R et al., Cancer Cell, 2016; Yang L et al., Cancer Cell, 2016). However, these findings have not been recapitulated using human tissue. The goals of this study were thus to determine the transcriptional and biological effects of DNMT3A mutations which contribute towards malignant transformation in human cells. Methods:To elucidate the effects of DNMT3A mutation, we introduced DNMT3A frameshift mutations into K562 cells using the CRISPR/Cas9 gene-editing system. We then performed various functional and genomic assays to better elucidate effects of DNMT3A loss. Results and Discussion:We successfully created 4 DNMT3A-KO K562 clones and 1 clone containing a mutation that produces an altered DNMT3A protein with an intact catalytic domain (DNMT3A-alt). We first assessed effects of DNMT3A loss on cell growth and apoptosis. DNMT3A-KO clones exhibited impaired growth compared to wild-type (WT) cells. DNMT3A-KO clones also displayed significantly increased apoptotic activity after exposure to 5-fluorouracil (5-FU). The DNMT3A-alt clone had similar growth and apoptotic activity to WT cells. We examined how DNMT3A loss impacted differentiation using phorbal 12-myristate 13-acetate (PMA), known to induce megakaryocytic differentiation of K562 cells. After overnight exposure to PMA, DNMT3A-KO clones exhibited less CD61 expression, a marker of megakaryocytic differentiation, than did WT cells. Again, the differentiation of the DNMT3A-alt clonewas comparable to WT. Finally, we performed karyotype analysis to elucidate a potential role of DNMT3A in maintaining genomic integrity. Surprisingly, DNMT3A-KO clones exhibited profound cytogenetic variability and genomic instability compared to WT, with most DNMT3A-KO clones containing dicentric chromosomes and ring forms in multiple spreads (Figure 1). The DNMT3A-alt clone had a karyotype identical to WT. CRISPR/Cas9-edited K562 clones without DNMT3A mutation (transfected WT or tWT) also had identical karyotypes to WT K562. TA cloning and mRNA sequencing were employed to elucidate whether loss of DNMT3A would lead to transcriptome instability. DNMT3A-KOand DNMT3A-altclones exhibited distorted splicing patterns, while tWT cell lines were comparable to WT. To further assess the effect of DNMT3A ablation on genomic integrity, we examined DNA-damage responses by measuring DNA double-stranded breaks (DSBs) after treatment with 5-FU. DNMT3A-KO clones were significantly more susceptible to DNA damage than were WT cells, while the DNMT3A-alt clone exhibited more DNA DSBs compared to WT only at high concentrations of 5-FU. Conclusion:CRISPR/Cas9-mediated DNMT3A-KO K562 cells may be used to model effects of DNMT3A mutations in human cells. Consistent with previous reports, our data suggest that DNMT3A is involved in the differentiation of multipotent progenitors. Novel to this approach, our findings implicate induction of genomic instability as a mechanism by which DNMT3A mutations might predispose to malignancy. Disclosures Hosokawa: Aplastic Anemia and MDS International Foundation: Research Funding. Townsley:Novartis: Research Funding. Young:GSK/Novartis: Research Funding.

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